Polyclonal antibody composition for treating allergy

ABSTRACT

A pharmaceutical composition for treating allergy is described. The composition comprises as an active ingredient a recombinant polyclonal antibody or a mixture of different monoclonal antibodies capable of reacting with or binding to an allergen together with one or more pharmaceutically acceptable excipients. The composition may be used topically as a solution, dispersion, powder, or in the form of microspheres. The polyclonal antibody is preferably a recombinant polyclonal antibody produced by phage display technology. The pairing of specific immunoglobulin variable region light chain and heavy chain maintained from the original polyclonal immune response or selected by panning using the allergen in question is preferably maintained by bulk transfer of the pairs into an expression vector.

FIELD OF INVENTION

The present invention relates to a composition comprising a recombinantpolyclonal antibody or a mixture of different monoclonal antibodies oran isolated or purified polyclonal antibody capable of reacting with orbinding to an allergen, as well as the use of a polyclonal antibodycapable of reacting with or binding to an allergen for the treatment ofallergy.

BACKGROUND OF THE INVENTION

The protective effects of humoral immunity are known to be mediated by afamily of structurally related glycoproteins called antibodies.Antibodies initiate their biological activity by binding to antigens.Antibody binding to antigens is generally specific for one antigen andthe binding is usually of high affinity. Antibodies are produced byB-lymphocytes. Blood contains many different antibodies, each derivedfrom a clone of B-cells and each having a distinct structure andspecificity for antigen. Antibodies are present on the surface ofB-lymphocytes, in the plasma, in interstitial fluid of the tissues andin secretory fluids such as saliva and mucus on mucosal surfaces.

All antibodies are similar in their overall structure, accounting forcertain similarities in physiochemical features such as charge andsolubility. All antibodies have a common core structure of two identicallight chains, each about 24 kilodaltons, and two identical heavy chainsof about 55-70 kilodaltons each. One light chain is attached to eachheavy chain, and the two heavy chains are attached to each other. Boththe light and heavy chains contain a series of repeating homologousunits, each of about 110 amino acid residues in length which foldindependently in a common globular motif, called an immunoglobulin (Ig)domain. The region of an antibody molecule formed by the association ofthe two heavy chains is hydrophobic. Antibodies are known to cleave atthe site where the light chain attaches to the heavy chain when they aresubjected to adverse physical or chemical conditions. Because antibodiescontain numerous cysteine residues, they have many cysteine-cysteinedisulfide bonds. All Ig domains contain two layers of beta-pleatedsheets with three or four strands of anti-parallel polypeptide chains.

Despite their overall similarity, antibody molecules can be divided intodistinct classes and subclasses based on physiochemical characteristicssuch as size, charge and solubility, and on their behavior in binding toantigens. In humans, the classes of antibody molecules are: IgA, IgD,IgE, IgG and IgM. Members of each class are said to be of the sameisotype. IgA and IgG isotypes are further sub-divided into subtypescalled IgA₁, IgA₂ and IgG₁, IgG₂, IgG₃ and IgG₄. The heavy chains of allantibody molecules in an isotype share extensive regions of amino acidsequence identity, but differ from antibodies belonging to otherisotypes or subtypes. Heavy chains are designated by the letters of theGreek alphabet corresponding to the overall isotype of the antibodymolecule, e.g., IgA contains α, IgD contains δ, IgE contains ε, IgGcontains γ, and IgM contains μ heavy chains. IgG, IgE and IgD circulateas monomers. IgA molecules secreted through the epithelia into themucosal lining of body cavities are homodimers whereas IgM molecules arepentamers. Circulating IgA exists mainly as a monomer. Multimeric formsof IgA and IgM are both stabilized by the so called J chain. SecretedIgA (S-IgA) is produced by B cells residing in lamina propria and takenup by epithelial cells on the basolateral side through thepoly-immunoglobulin receptor (pIgR), transported through the epithelialcell and secreted into the mucosa on the luminal side. When the IgA:Jchain:pIgR complex is released, the pIgR is cleaved by a protease and apart of the pIgR molecule called the secretory component (SC) remainsbound to the IgA:J chain complex. Thus, S-IgA is a complex consisting ofIgA, the J chain, and the SC of which the two latter are covalentlybound to the IgA molecule through disulphide bonds. S-IgA is veryresistant to the proteolytic environment of the epithelial mucosa e.g.in the respiratory or the gastrointestinal tract, and as such make upthe primary specific immune system in these sites. It has beendemonstrated that S-IgA has an immunomodulating effect and may inducetolerance to the antigens they bind.

There are between 10⁸ and 10¹⁰ structurally different antibody moleculesin every individual, each with a unique amino acid sequence in theirantigen combining sites. Sequence diversity in antibodies ispredominantly found in three short stretches within the amino terminaldomains of the heavy and light chains called variable (V) regions, todistinguish them from the more conserved constant (C) regions.

Immunoglobulin E (IgE) is responsible for so-called type 1hypersensitivity which manifest itself as common diseases such asallergic rhinitis, allergic conjunctivitis, hay fever, allergic(extrinsic) asthma, bee venom allergy, and food allergy.Allergen-specific IgE is produced in excess in patients withIgE-mediated allergies. IgE circulate in the blood and bind tohigh-affinity Fc receptors for IgE on basophils and mast cells in blood,various tissues, or on mucosal surfaces. In most allergic responses, theallergens enter the body of a patient through inhalation, ingestion, orthrough the skin. The allergen molecules bind to preformed IgE alreadybound to the high-affinity receptor FcεRI on the surfaces of mast cellsand basophils, resulting in the crosslinking of several IgE moleculesand triggering the release of histamine and other inflammatory mediatorscausing the various allergic symptoms.

Among the tissues that are most susceptible to local IgE-mediatedallergic reactions are the conjunctiva, the mucosa of the nasal cavityor the oropharynx (allergic rhinitis), the mucosal linings of thebronchial tract, and the gastrointestinal mucosa. Thus, allergens enterthe respiratory tract through inhalation and get trapped on the mucosalsurfaces of the nasal lining or the bronchial passages of therespiratory tract. Airborne allergens also get in contact with moistsurfaces of eyes and ears and are retained on the mucosa. The mucosaltissues are densely populated with mast cells and allergens arriving atthese sites may therefore bind IgE and activate mast cells.

The therapeutic principles and treatment modalities in the management ofallergy have not changed substantially in recent years.Immunosuppressive drugs such as steroids for suppressing immuneactivities and bronchial dilators for relieving asthma symptoms havelong been the main treatment modality for patients with allergic asthma.Desensitization immunotherapy is the most important novel therapy forseverely affected patients, but the medical advances have been limitedto refining the classification of the allergenic substances, improvingdiagnostic methods, and providing a better controlled and broaderlibrary of allergen extracts for immunotherapy. As for research,progress has been made in the identification and isolation of majorallergenic components of allergenic substances. For example, the majorallergic components of ragweed, house dust mites, and cat and dog danderand saliva have been identified. When the allergen particles, e.g.timothy grass pollen arrive to the airway mucosa they disintegrate intomajor and minor allergic components.

Antibodies have been suggested for a number of clinical treatments:MedImmune Inc. is studying the use of humanized anti-respiratorysyncytial virus (RSV) monoclonal antibodies and markets a polyclonalanti-RSV human immunoglobulin product (RespiGam) isolated from humandonor blood and used to treat RSV infection. MedImmune also marketsCytoGam, an anti-CMV (cytomegalovirus) human immunoglobulin for thetreatment of CMV infection. IDEC and Genentech are jointly performingclinical trials of a chimeric mouse-human monoclonal antibody(Rituximab) aimed at the CD20 antigen found on mature B cells and mostnon-Hodgkin's lymphoma tumors for the treatment of relapsed orrefractory low-grade non-Hodgkin's lymphoma. GalaGen is studying the useof bovine polyclonal immunoglobulin (Diffistat-G) for treatment ofClostridium difficile antibiotic associated diarrhea. SmithKline Beechamand Schering-Plough are developing an anti-IL-5 monoclonal antibodywhich has been shown in clinical trials to prevent eosinophilicinflammation and airway constriction. An anti-IgE monoclonal antibody isbeing developed by Genentech to “switch-off” allergies. The antibodyRhu-Mab-E25, which is a humanized chimeric IgG₁ monoclonal antibodyspecific for a unique epitope on human high affinity IgE receptors(FcεRI), has been shown to reduce free IgE levels after the firstadministration by injection. It attenuated both early and late phaseresponses to inhaled allergens after multiple injections. Examples ofantibodies used therapeutically also include a nebulized IgG (Sandoz),which is used intranasally against RSV; HNK20 (Oravax), an anti-RSV IgA;and 4B9 (Bristol Myers-Squibb), an anti-group B Streptococcus IgMmonoclonal antibody. Other therapeutically useful monoclonal antibodiesinclude monoclonal anti-CD4 antibodies, anti-IL-2 antibodies andanti-IL-4 antibodies.

The immunotherapy of RSV infection using small particle aerosols of IgGhas been disclosed by Piazza et al. (J. Infect. Dis., Vol. 166, pp.1422-1424, 1992). In this study it was shown that a 15-minute exposureto an aerosolized 5% solution of IgG effected a 50-fold reduction inpulmonary virus. Brown (Aerosol Science and Technology, Vol. 24, pp.45-56, 1996) discloses the use of antibodies as in hibitors orantagonists of cytokines to depress respiratory inflammatory diseases orallergen-induced asthmatic responses. Also mentioned is localrespiratory delivery of pathogen-specific antibody for treatment ofacute viral or bacterial respiratory infections.

Antibody liposomes, i.e., immunoliposomes, are disclosed by Maruyama etal. (Biochim. Biophys. Acta, Vol. 1234, pp. 74-80, 1995). Coatingliposomes with antibody leads to enhanced uptake of the liposome by thereticuloendothelial system. Human monoclonal antibodies are known to beuseful as anti-tumor agents. A mouse/human monoclonal IgG antibodyspecific for the Lewis Y antigen found on the surface of tumor cells isdisclosed by Paborji et al. (Pharmaceutical Research, Vol. 11, No. 5,pp. 764-771, 1994). The use of antibodies in metered-dose propellantdriven aerosols for passive antibody aerosol therapy against respiratoryinfections is suggested in Brown et al. (Journal of ImmunologicalMethods, Vol. 176, pp. 203-212, 1994). Immune responses in therespiratory tract are of great importance for protection againstinfections of the respiratory system and for their involvement inrespiratory allergies and asthma. Effective targeting ofimmunomodulating reagents including monoclonal antibodies to therespiratory tract is shown to be of benefit in increasing local immunityto respiratory pathogens or decreasing immune-mediated respiratorypathology. Inhaled immunoconjugates, immunoliposomes orimmunomicrospheres have application in the lung as killers of cancercells (immunoconjugates) or, in the case of immunoliposomes andmicrospheres, as stealth delivery particles of a variety of therapeuticagents. An IgM anti-group B Streptococcus monoclonal antibody isdisclosed by Gombotz et al. (Pharmaceutical Research, Vol. 11, pp.624-632, 1994).

U.S. Pat. No. 5,670,626 proposes the use of monoclonal antibodies forthe treatment of IgE-mediated allergic diseases such as allergicrhinitis, allergic asthma and allergic conjunctivitis by employingmonoclonal antibodies to inhibit the entry of allergenic molecules intomucosal tissues. The binding of allergenic molecules by antibodies isassumed to inhibit the allergens from being taken up by mucosalepithelial cells.

In certain clinical situations, the use of monoclonal antibodies isassociated with specific disadvantages. Thus, monoclonal antibodies aredirected against single antigenic epitopes. Therefore, if the target isof a complex nature presenting many different epitopes then thefunctional avidity of the monoclonal antibody may be low or loweredbelow a critical threshold allowing the target to escape eliminationthrough immune recognition.

Also, because monoclonal antibodies are directed against singleantigenic determinants, the density of the antibody targets on e.g.allergens may not be high enough to mediate elimination of the allergen.The efficient activation of complement similarly requires high targetantibody densities which may not be achieved with single specificitymonoclonal antibodies.

Thus, in the case of allergens, monoclonal antibodies are sub-optimal asthey are directed against single epitopes. The majority of allergens arecomplex proteins, consisting of many protein and peptide epitopes, andexisting in many variants. Thus, a single monoclonal antibodypreparation cannot be expected to exhaustively cover more than aminority of the possible epitopes on an allergen, e.g. a pollen particleor proteins from cat dander. This means that if the desired clinicaleffect of an antibody can be characterized as a complete blocking of theavailable antibody epitopes, then a single monoclonal antibody will notbe sufficient. Further, if an antibody preparation should preferably bedeveloped against several homologous allergens from closely relatedallergens, e.g. pollens, or against several proteins from one allergensource e.g. animal dander, then a single monoclonal antibody will notmeet the required efficacy.

Nevertheless, a paper by Schwarze and coworkers (Am. J. Resp. Crit. CareMed., Vol. 158, pp. 519-525, 1998) investigated the therapeutic efficacyof a monoclonal antibody directed against the major ragweed allergen Amba/in a murine allergy model based on mice (Balb/c) sensitized andchallenged with both Amb a/and whole ragweed extracts. It wasdemonstrated that administration of the monoclonal IgA antibody beforeallergen exposure decreased airway responsiveness to metacholinechallenge, and decreased the number of pulmonary eosinophils and Amba/-specific IgE levels in serum. Moreover, the study indicate thatadministration of IgA had an immunomodulatory effect implying that IgAtreatment could have a long-term desensitizing effect on allergy.However, it must be stressed that this allergen model is based on theinduction of allergy-like symptoms using a single allergen, Amb a I.Thus, the study does not take into account that the vast majority ofallergies are caused by reactions towards a number of allergen proteinsand epitopes derived from a single allergen particle, which emphasizesthe need for a polyclonal antibody mixture in this regime of treatment.Furthermore, human allergy is profoundly more complex than theallergy-like symptoms induced in an inbred mouse strain (Inhal.Toxicol., Vol 12, pp. 829-622, 2000). Consequently, the potentialusefulness of monoclonal antibodies as allergen blocking agents islimited. Finally, monoclonal antibodies may display cross-reactivity toantigenic structures of host cell tissue resulting in potential unwantedside effects. When this occurs the cross-reactivity cannot be removed byadsorption. Therefore a large number of different monoclonal antibodiesmay need to be produced in order to generate the desired combination ofantigen specificity and target selectivity, and even so there stillremains a significant risk of cross-reactivity towards endogenousself-antigens in a proportion of patients.

A separate issue is the generation of human anti-mouse antibodyresponses (HAMA). Conventional murine monoclonal antibodies are foreignproteins to the human recipient, and therefore a HAMA immune response isoften elicited in the recipient, which may lead to unwanted side effectsin addition to reduced treatment efficacy. In order to circumvent thisproblem, chimeric monoclonal antibodies possessing human constant (C)regions and murine variable (V) regions have been developed. Furthermorehumanized monoclonal antibodies, where only the hypervariablecomplementarity determining region (CDR) is derived from mousemonoclonal antibodies and finally, so-called fully human monoclonalantibodies produced in mice transgenic for human immunoglobulin geneshave been developed to avoid these problems. However, a potential forthe generation of anti-idiotype antibody responses specific for theV-region specificity determining CDR still exists when injecting largeamounts of monoclonal antibodies with identical V-regions.

For these reasons as outlined above, it may often be preferable to usepolyclonal antibodies.

In WO 98/10776 it is theorized that phospholipase A₂ (PLA₂) is involvedin the pathogenesis of many diseases acting as an inflammatory mediatorpromoting chronic inflammation. Thus it is suggested to use serumreactive with at least one phospholipase A₂ enzyme for the treatment ofneoplasms in mammals. There is no suggestion to use polyclonalantibodies for blocking the uptake of an allergen by topicaladministration of an antibody binding to the allergen.

U.S. Pat. No. 4,740,371 describes a modification of allergenimmunotherapy whereby an immune complex of the allergen and an antibodythereto is used for desensitization treatment, the antibody beingpresent in molar excess with respect to the allergen to prevent ananaphylactic response. The purpose of the inclusion of the antibody inthis treatment is to decrease the risk of allergic side effects such asanaphylactic shock to the desensitization treatment. The proportion ofantibody to be added to the allergen is defined essentially by theneutralizing power of the antibody. Enough antibody must be used so thatwhen the composition is administered, there is practically no allergiceffect induced by the allergen. The adding of antibody to the allergencomposition is solely a remedy to avoid side effects of the allergenexposure, the treatment still being an allergen immunotherapy.

There are several drawbacks of using conventional polyclonal antibodiesin the treatment of allergy. First of all, polyclonal antibodies in theform of IgG purified from hyperimmune human serum is available inlimited supply and in amounts insufficient for the treatment of allergicdiseases and other common conditions. Also, gamma globulin preparationsare expensive to produce, and display low efficacy due to their mixednature containing an overwhelming majority of non-specific human serumimmunoglobulin reactivities. Also, there exist a real risk oftransmitting contaminating reagents, including infectious microorganisms(hepatitis virus, HIV, prions, others), or mitogens, cytokines andtoxins. Finally, the variability between preparations remains a majorproblem. In order to solve the problem of supply, xenogeneic sources ofpolyclonal antibodies including serum from immunized non-human animalshave been tested. However, such compositions may result in thegeneration of potent anti-xenoantibody responses, and carries a realrisk of serious side effects such as anaphylactic shock or serumsickness, as well as the transmission of xenotropic infections.

U.S. Pat. No. 5,789,208 describes the use of a recombinant polyclonalantibody for vaccine therapy and prophylaxis to treat or preventneoplastic diseases. The antibodies are used for boosting a patient'simmune system for the possible later recognition of the antigen to whichthe antibody binds and thereby initiate an elimination reaction. Thevaccination will have to be repeated to be effective. There is nosuggestion to use polyclonal antibodies reacting with or binding toallergens in allergy treatment where the polyclonal antibodies should beadministered completely differently before, during, or shortly after thepatient has been exposed to an allergen.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a pharmaceuticalcomposition comprising as an active ingredient a recombinant polyclonalantibody or a mixture of different monoclonal antibodies or an isolatedor purified polyclonal antibody capable of reacting with or binding toan allergen together with one or more pharmaceutically acceptableexcipients.

In most embodiments, the pharmaceutical composition according to theinvention is free of the allergen to which the recombinant polyclonalantibody or the mixture of different monoclonal antibodies or theisolated or purified polyclonal antibody bind or is reactive with.However, in special cases during so-called specific allergenimmunotherapy to induce allergen tolerance in a patient, theallergen-specific polyclonal antibody may be administered to the patientin conjunction with an allergen preparation, in order to enable orenhance the efficacy of the tolerance induction procedure.

In another aspect, the invention relates to the use of a polyclonalantibody with desired specificities for the manufacture of apharmaceutical composition for the prophylaxis or treatment of allergyor allergic conditions such as allergic rhinitis, allergicconjunctivitis, hay fever, asthma, etc.

In a further aspect, the invention relates to a method of preventing ortreating allergy, which comprises administering to a patient in needthereof a sufficient amount of a polyclonal antibody capable of reactingwith or binding to an allergen to which the patient has shown anallergic reaction.

The use of a polyclonal antibody has potential clinical advantagescompared with the use of a monoclonal antibody due to the presence ofmultiple reactivities in a polyclonal antibody against theallergen-target in question. There may be generated a polyclonalantibody which has reactivities against all epitopes on a complexallergen target. Due to the polyclonal nature of the composition,containing many epitope specificities, the functional antibody densitywhich can be achieved on complex allergen antigens when using apolyclonal antibody is significantly higher, than with a monoclonalantibody. This results in more efficient blocking or clearance of thetarget allergen. Further, the polyclonal nature of the compositionenables recognition of and blocking of epitopes on related, homologousallergen isotypes, due to broad reactivity with several epitopes sharedin part between related allergens, something which is not enabled by amonoclonal antibody.

Further, it can be expected that treatment with allergen-specificpolyclonal antibodies of the IgA or IgG isotype will have animmunomodulating effect by inducing tolerance to an allergen, and thushave a long term effect in curing the allergy or reducing the need forfurther treatment. Thus, a further aspect of the invention relates tothe use of a pharmaceutical composition according to the invention forprophylactic treatment inducing tolerance to the allergen. This may evenbe used in patients where an allergic reaction has not yet been observedbut which patients due to family history or genetic analysis are likelyto develop allergy to an allergen.

Furthermore, contrary to a monoclonal antibody, a polyclonal antibodypreparation comprises a mixture of specificities, and therefore anysingle and individual, cross-reacting specificity idiotype will bedelivered at a very low concentration, thus reducing significantly thepotential for harmful side-effects, due to cross-reactivity. In otherwords, the potential for deleterious side-effects due to unwanted tissuecross-reactivity is diluted out in the polyclonal antibody reagent.Further, any unwanted cross-reactivity of the polyclonal antibodypreparation can be removed by adsorption. If a monoclonal antibodyresults in an unwanted cross-reactivity, it is inherent to the singleantibody present and can of course not be removed without destroying theactivity of the preparation.

Also, in analogy with the properties of polyclonal antibodies in termsof the diminished potential for cross-reactivity, polyclonal antibodieswill also be much less likely than monoclonal antibodies to induce aneutralizing anti-idiotype immune response, since each singleepitope-specific idiotype of the administered polyclonal antibodypreparation is present in a very low quantity or concentration, beingbelow the threshold for generation of an anti-idiotype response.

Some of the drawbacks of using conventional polyclonal antibodies in theform of IgG purified from hyperimmune human (limited supply, expensiveto produce) or serum from normal animals (anti-xeno-antibody responses,anaphylactic shock) is the use of serum or other biological materialfrom animals transgenic for human immunoglobulin genes. Thus, suchanimals can be immunized with allergens, and used as a source to isolateallergen-specific polyclonal antibody products of fully human sequence.

The immune complexes on the nasal linings will be cleared as the mucousexcretion is swallowed. The immune complexes on the mucosal surfaces ofthe tracheal and bronchial airways will be expelled into the mouth,mixed with saliva, swallowed and digested in the gastrointestinal tract.In order to achieve betters effects in adsorbing and clearing allergenicmolecules from the mucous fluids on the mucosal surfaces and preventingany uptake of the complexed allergen by the mucosal epithelial cells,the allergen-specific antibody can be conjugated to polymer backbones ormicrobeads forming microspheres.

Thus the pharmaceutical composition according to the invention may beformulated as a solution, dispersion, powder, or in form ofmicrospheres.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody molecule” describes the single antibody proteinmolecule or fragments thereof containing one or more variable antigenbinding domain(s) and constant regions. An antibody molecule is usuallymonospecific, but may also be described as idiospecific, heterospecific,polyspecific or of unwanted specificity. It cannot be non-specificexcept in the sense of non-immunochemical binding. Antibody moleculesbind by means of specific binding sites to specific antigenicdeterminants or epitopes on antigens.

Collectively, antibodies may exist as a population of molecules where afraction or all of the members are capable of reacting with a specificantigen determinant. Thus, in the present context, the term “antibody”refers to compositions/mixtures/populations of antibody molecules, suchas they are found as the functional component of anti-serum or immuneserum derived from mammals, or as they are found in monoclonal orpolyclonal antibody compositions with similar functionality preparedeither from human or animal sources or by recombinant technologies,including transgenic animals and phage display or by conventionalhybridoma technology.

The term “polyclonal antibody” denotes a mixture of different antibodymolecules which react with more than one immunogenic determinant of anantigen.

In the present context, the term “polyclonal antibody” encompasses apolyclonal antibody isolated or purified from mammalian blood,secretions, or other fluids, or from eggs, as well as a mixture ofdifferent monoclonal antibodies, and finally a polyclonal antibody maybe produced as a recombinant polyclonal antibody.

The term “recombinant polyclonal antibody” refers to a polyclonalantibody generated by the use of recombinant technologies, and suchpolyclonal antibodies are hereafter named symphobodies. Thus, asymphobody contains a high concentration of different antibodymolecules, all or a majority of which are displaying a desired bindingactivity towards an antigen composed of more than one epitope.

Symphobodies can be generated by recombinant DNA techniques followed byexpression in eukaryotic cells, including yeast, fungi, insect, plant,or mammalian cells, or in prokaryotic cells such as bacteria, or asexpressed from virus vectors, or through gene therapy, or fromexpression of transgenes in animals.

Preferably at least 85% of the antibody molecules in the symphobodypreparation are target-specific, more preferably at least 90% aretarget-specific, even more preferably at least 95% are target-specific,and most preferably all antibody molecules in the symphobody preparationare target-specific.

By the term “a mixture of different monoclonal antibodies” is meant amixture of two or more different monoclonal antibodies. The term “two ormore” in the present context denotes from 2 to 100, preferably from 3 to60, more preferably from 5 to 40, most preferably from 10 to 25different monoclonal antibodies.

By the term “an isolated or purified polyclonal antibody” is meant apolyclonal antibody isolated or purified from mammalian blood,secretions, or other fluids, or from eggs.

It is to be understood that the expressions “an antibody, a polyclonalantibody, a recombinant antibody, a mixture of different monoclonalantibodies and an isolated or purified polyclonal antibody” all alsoencompasses functional fragments of the mentioned antibodies.

A currently preferred method of preparing a recombinant polyclonalantibody is by making polyclonal antibody libraries (PCAL), for instanceas disclosed in U.S. Pat. No. 255,789,208 (to J. Sharon) which is herebyincorporated by reference in its entirety.

More specifically, the polyclonal antibody included in thepharmaceutical composition may be prepared by immunizing an animal,preferably a mammal, with an allergen of choice followed by theisolation of antibody-producing B-lymphocytes from blood, bone marrow,lymph nodes, or spleen. Alternatively, antibody-producing cells may beisolated from an animal and exposed to an allergen in vitro againstwhich antibodies are to be raised. The antibody-producing cells may thenbe cultured to obtain a population of antibody-producing cells,optionally after fusion to an immortalized cell line such as a myeloma.

More preferably, as a starting material B-lymphocytes may be isolatedfrom the tissue of an allergic patient, in order to generate fully humanpolyclonal antibodies.

The present composition may also be generated using suitable tissue frommice, rats, pigs (swine), sheep, bovine material, or other animalstransgenic for the human immunoglobulin genes, as starting material inorder to generate fully human polyclonal antibodies.

Particularly, in the case of mice or other animals transgenic for thehuman immunoglobulin genes (e.g. as disclosed in U.S. Pat. No.5,939,598), the animals may be immunized to stimulate the in vivogeneration of specific antibodies and antibody producing cells beforepreparation of the polyclonal antibody composition from the animal byextraction of B lymphocytes or purification of polyclonal serum.

A combinatorial library may be prepared from immunized B lymphocytes byassociating V_(L) and V_(H) randomly in a cloning vector. Thus, therecombinant polyclonal antibody is generated under such conditions thatthe immunoglobulin heavy chain variable region and light chain variableregion gene segments are linked together randomly in order to allow forthe bulk transfer of variable region light chain and heavy chain genepairs from one vector to another, while allowing stable pairing ofspecific immunoglobulin variable region light chain and heavy chain genesegments as they are present upon selection from a parental library ofimmunoglobulin variable region light chain and heavy chain gene segmentpairs encoding antibody molecules capable of reacting with or binding toan allergen.

Single cell PCR may be used in an attempt to retain the native pairingof V_(L) and V_(H) in the single cell. In this case antibody-producingB-lymphocytes which have been isolated from animals or humans may befixed with a fixative solution or a solution containing a chemical suchas formaldehyde, glutaraldehyde or the like.

The cells are then permeabilized with a permeabilization solutioncomprising for example a detergent such as Brij, Tween, polysorbate,Triton X-100, or the like. The fixing and permeabilization processshould provide sufficient porosity to allow entrance of enzymes,nucleotides and other reagents into the cells without undue destructionof cellular compartments or nucleic acids therein. Addition of enzymesand nucleotides may then enter the cells to reverse transcribe cellularV_(H) and V_(L) mRNA into the corresponding cDNA sequences.

Reverse transcription may be performed in a single step or optionallytogether with a PCR procedure, using a reverse transcriptase, sufficientquantities of the four dNTPs and primers that bind to the mRNA providinga 3′ hydroxyl group for reverse transcriptase to initiatepolymerization. Any primer complementary to the mRNA may be used, but itis preferred to use primers complementary to the 3′-terminal end of theV_(H) and V_(L) molecules so as to facilitate selection of variableregion mRNA.

Upon reverse transcription, the resulting cDNA sequences may beamplified by PCR using primers specific for immunoglobulin genes and, inparticular, for the terminal regions of the V_(H) and V_(L) nucleicacids. PCR procedures may be followed as disclosed in, e.g., U.S. Pat.No. 4,683,195. Preferably, the cDNAs are PCR amplified and linked in thesame reaction, using, in addition to the cDNA primers, one primer forthe 5′ end of the V_(H) region gene and another for the 5′ end of theV_(L) gene. These primers also contain complementary tails of extrasequence, to allow the self-assembly of the V_(H) and V_(L) genes. AfterPCR amplification and linking, the chance of getting mixed products, inother words, mixed variable regions, is minimal because theamplification and linking reactions were performed within each cell. Therisk of mixing can be further decreased by utilizing bulky reagents suchas digoxigenin labeled nucleotides to further ensure that V region cDNApairs do not leave the cellular compartment and intermix, but remainwithin the cell for PCR amplification and linking. The amplifiedsequences are linked by hybridization of complementary terminalsequences. After linking, sequences may be recovered from cells. Forexample, after linking, cells can be washed in a solution of sodiumdodecyl sulfate (SDS). The SDS precipitates out of the cells afterincubation on ice and the supernatant can be electrophoresed into anagarose or acrylamide gel. Alternatively, or in combination with the SDSprocess, using a reagent such as digoxigenin-linked nucleotides, DNAproducts synthesized will remain within the cell and be amplified. Thelinked product is recovered upon electrophoresis of the supernatant.

After electrophoresis of the supernatant, the gel slice corresponding tothe appropriate molecular weight of the linked product is removed andthe DNA isolated on, for example, silica beads. The recovered DNA can bePCR amplified using terminal primers, if necessary, and cloned intovectors which may be plasmids, phages, cosmids, phagemids, viral vectorsor combinations thereof. Convenient restriction enzyme sites may beincorporated into the hybridized sequences to facilitate cloning. Thesevectors may also be saved as a library of linked variable regions forlater use.

The linked V_(H) and V_(L) region genes may be PCR amplified a secondtime using terminal nested primers, yielding a population of DNAfragments which encode the linked V_(H) and V_(L) genetic regions. Thegrouping of V_(H) and V_(L) combinations is an advantage of this processand allows for the in mass or batch transfer of all clones and all DNAfragments during this and all cloning procedures.

Preferably, the recombinant polyclonal antibody may be generated undersuch conditions that the immunoglobulin heavy chain variable region andlight chain variable region gene segments are linked together in ahead-to head orientation, in order to allow for the bulk transfer ofvariable region light chain and heavy chain pairs from one vector toanother, including from phage to vector, and including from the cell oforigin to phage or vector, resulting in a stable pairing of specificimmunoglobulin variable region light chain and heavy chains genesegments as they are found in the original polyclonal immune response ofthe animal or human individual.

It may sometimes be desirable to treat the variable region genesequences with a mutating agent. Mutating agents create point mutations,gaps, deletions or additions in the genetic sequence which may begeneral or specific, or random or site directed. Useful mutating agentsinclude ultraviolet light, gamma irradiation, chemicals such as ethidiumbromide, psoralen and nucleic acid analogs, or DNA modifying enzymessuch as restriction enzymes, transferases, ligases and specific andnonspecific nucleases and polymerases. Moreover it may be feasible touse mutator strains. In particular, random mutations may be introducedin the CDRs of the V_(H) and V_(L) region genes by oligonucleotidedirected mutagenesis. Mutations introduced into the gene sequence willultimately increase library complexity and diversity as well as affinityfor antigen which may further increase the library's usefulness intreatment. Furthermore, such mutagenesis may be used on a single V_(H)and V_(L) pair or on a defined group of such pairs to generate a libraryde novo.

Cloning is performed, for example, by cleaving the cDNA and vectorsequences with a restriction enzyme, if necessary isolating certainnucleic acid fragments, mixing the fragments together in the presence ofligase in a suitable balanced salt solution, and incubating the mixtureunder enzymatically acceptable conditions for a prescribed period oftime. Using different enzyme recognition sites at each terminus of thecDNA, cloning orientation can be predetermined.

Vectors are transformed into suitable host cells and the culturesamplified to expand the different populations of vectors that comprisethe library. Host cells for prokaryotic vectors may be a culture ofbacteria such as Escherichia coli. Host cells for eukaryotic vectors maybe a culture of eukaryotic cells such as any mammalian, insect or yeastcell lines adapted to tissue culture. Bacterial cells are transformedwith vectors by calcium chloride-heat shock or electroporation, althoughmany other transformation procedures would also be acceptable.Eukaryotic cells are transfected with calcium phosphate precipitation orelectroporation, although many other transformation procedures wouldalso be acceptable. The DNA fragments may be cloned into prokaryotic oreukaryotic expression vectors, chimeric vectors or dual vectors. Theexpression vector may be a plasmid, cosmid, phage, viral vector,phagemid and combinations thereof, but is preferably a phage displayvector wherein the recombinant product is expressed on the phage surfaceto facilitate screening and selection. Useful transcriptional andtranslational sites may be placed on the expression vector including RNApolymerase recognition regions such as a TATA box site, a CAT site, anenhancer, appropriate splicing sites, if necessary, a AT rich terminalregion and a transcription initiation site. Useful sites to facilitatetranslation include translational start and stop sites and ribosomebinding sites. Typically, some of the more useful sites for efficienteukaryotic expression, such as the SV40, CMV, HSV or baculoviruspromoter/enhancer region, are derived from viruses. The resultingrecombinant antibody may be of the murine class IgG₁, IgG_(2a),IgG_(2b), IgM, IgA, IgD or IgE, the human classes IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, IgA₂, IgA₂, IgD or IgE, or combinations or fragmentsthereof. Preferably, the chimeric antibody library is composed ofprimarily IgG antibodies or Fab antibody fragments.

Selection of a recombinant polyclonal antibody with desired specificitycan be performed e.g. by affinity selection (panning) using anallergen-coated surface for binding the phage particles exhibiting arelevant antibody specificity. The majority of phages in the phagelibrary are eliminated by washing and the bound phage particles areretrieved by harsher conditions (elution). After the selectionprocedures, the V_(L) and V_(H) antibody gene pairs in the selectedlibrary of phage particles can be subcloned into a different vectordesigned for expression of the recombinant polyclonal antibody as acomplete antibody molecule or a fragment thereof such as a Fab fragment.

The use of recombinant DNA technology for generating a recombinantpolyclonal antibody is a cost-effective way of generating antibodies,and the production of well-characterized, polyclonal antibodypreparations with desired specificities, would overcome the aboveproblems with conventional polyclonal antibody sera and individualmonoclonal antibodies and allow the use of such reagents for theprophylaxis or treatment of allergy or allergic conditions, e.g. asthma.

Pharmaceutical Compositions

In a preferred embodiment, the pharmaceutical composition of theinvention is one intended for topical administration/application tomucosa, such as the oropharynx, nasal cavity, respiratory tract,gastrointestinal tract, eye such as the conjunctival mucosa, vagina,urogenital mucosa, or for dermal application.

A particularly interesting use of the pharmaceutical composition is forapplication to the nasal, bronchial or pulmonary mucosa. Specifically,the topical treatment of allergy using inhaled polyclonal antibodieswould be a particularly useful application of such reagents, allowingthe discovery and development of novel therapeutic or preventivemodalities which are cheap to produce, harmless and of no toxicity, andaimed towards a disease afflicting a very large proportion of the humanpopulation.

In order to obtain optimal delivery of the polyclonal antibody to thepulmonary cavity in particular, it may be advantageous to add asurfactant such as a phosphoglyceride, e.g. phosphatidylcholine, and/ora hydrophilic or hydrophobic complex of a positively or negativelycharged excipient and a charged antibody of the opposite charge.

Other excipients suitable for pharmaceutical compositions intended fordelivery of the polyclonal antibody to the respiratory tract mucosa maybe from the group consisting of a) carbohydrates, e.g., monosaccharidessuch as fructose, galactose, glucose, D-mannose, sorbiose, and the like;disaccharides, such as lactose, trehalose, cellobiose, and the like;cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin; andpolysaccharides, such as raffinose, maltodextrins, dextrans, and thelike; b) amino acids, such as glycine, arginine, aspartic acid, glutamicacid, cysteine, lysine and the like; c) organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate,magnesium gluconate, sodium gluconate, tromethamine hydrochloride, andthe like; d) peptides and proteins, such as aspartame, human serumalbumin, gelatin, and the like; e) alditols, such as mannitol, xylitol,and the like, f) polycationic polymers, such as chitosan or a chitosansalt or derivative.

Over the years certain drugs have been sold in compositions suitable forforming a drug dispersion for oral inhalation (pulmonary delivery) totreat various conditions in humans. Such pulmonary drug deliverycompositions are designed to be delivered by inhalation by the patientof the drug dispersion so that the active drug within the dispersion canreach the lung.

Pulmonary drug delivery can itself be achieved by different approaches,including liquid nebulizers, aerosol-based metered-dose inhalers (MDI's)and dry powdered dispersion devices. Chlorofluorocarbon (CFC) basedMDI's are losing favor because of their adverse effect on theenvironment. Dry powder dispersion devices, which do not rely on CFCaerosol technology, are promising for delivering drugs that may bereadily formulated as dry powders. Many otherwise labile macromoleculesmay be stably stored as lyophilized or spray dried powders, either bythemselves or in combination with suitable powder carriers.

Many pharmaceutical compositions, including antibodies, are quiteexpensive. Thus, the ability to efficiently formulate, process, packageand deliver the dry powders with minimal loss of drug is critical.

An important requirement for hand held and other powder delivery devicesis efficiency. It is important that the delivered dose be relativelyhigh to reduce the number of breaths required to achieve a total dosage.The ability to achieve both adequate dispersion and small dispersedvolumes is a significant technical challenge that requires in part thateach unit dosage of the powder composition be readily and reliablydispersible. Certain pulmonary delivery devices, such as those disclosedin U.S. Pat. No. 5,797,392, U.S. Pat. No. 5,458,135 and InternationalPatent Publication WO96/09085 are useful for pulmonary delivery of drypowder drugs. Other administration forms of the present compositioninclude liquids, gels, ointments or other suitable formulations forocular administration, sprays, aerosols, powders, or other compositionsfor the administration into the nasal cavity, chewing gum, pasta orother compositions for oral cavity, creams, ointments, lotions, gels orother compositions suitable for the application onto the skin,vagitories, gels or other compositions suitable for application onto thevaginal or urogenital mucosa or formulated as capsules or tablets forthe administration into the digestive tract. For dermal application, thepolyclonal antibody may suitably be formulated with one or more of thefollowing excipients: solvents, buffering agents, preservatives,humectants, chelating agents, antioxidants, stabilizers, emulsifyingagents, suspending agents, gel-forming agents, ointment bases,penetration enhancers, perfumes, and skin protective agents.

Examples of solvents are e.g. water, alcohols, vegetable or marine oils(e.g. edible oils like almond oil, castor oil, cacao butter, coconutoil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanutoil, poppyseed oil, rapeseed oil, sesame oil, soybean oil, sunfloweroil, and tea seed oil), mineral oils, fatty oils, liquid paraffin,polyethylene glycols, propylene glycols, glycerol, liquidpolyalkylsiloxanes, and mixtures thereof.

Examples of buffering agents are e.g. citric acid, acetic acid, tartaricacid, lactic acid, hydrogenphosphoric acid, diethyl amine etc.

Suitable examples of preservatives for use in compositions areparabenes, such as methyl, ethyl, propyl p-hydroxybenzoate,butylparaben, isobutylparaben, isopropylparaben, potassium sorbate,sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol, bronopol,bronidox, MDM hydantoin, iodopropynyl butylcarbamate, EDTA, benzalconiumchloride, and benzylalcohol, or mixtures of preservatives. Examples ofhumectants are glycerin, propylene glycol, sorbitol, lactic acid, urea,and mixtures thereof.

Examples of antioxidants are butylated hydroxy anisole (BHA), ascorbicacid and derivatives thereof, tocopherol and derivatives thereof,cysteine, and mixtures thereof. Examples of emulsifying agents arenaturally occurring gums, e.g. gum acacia or gum tragacanth; naturallyoccurring phosphatides, e.g. soybean lecithin; sorbitan monooleatederivatives; wool fats; wool alcohols; sorbitan esters; monoglycerides;fatty alcohols;, fatty acid esters (e.g. triglycerides of fatty acids);and mixtures thereof.

Examples of suspending agents are e.g. celluloses and cellulosederivatives such as, e.g., carboxymethyl cellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carraghenan, acacia gum, arabic gum,tragacanth, and mixtures thereof.

Examples of gel bases, viscosity-increasing agents or components whichare able to take up exudate from a wound are: liquid paraffin,polyethylene, fatty oils, colloidal silica or aluminum, zinc soaps,glycerol, propylene glycol, tragacanth, carboxyvinyl polymers,magnesium-aluminum silicates, Carbopol®, hydrophilic polymers such as,e.g. starch or cellulose derivatives such as, e.g.,carboxymethylcellulose, hydroxyethylcellulose and other cellulosederivatives, water-swellable hydrocolloids, carragenans, hyaluronates(e.g. hyaluronate gel optionally containing sodium chloride), andalginates including propylene glycol alginate.

Examples of ointment bases are e.g. beeswax, paraffin, cetanol, cetylpalmitate, vegetable oils, sorbitan esters of fatty acids (Span),polyethylene glycols, and condensation products between sorbitan estersof fatty acids and ethylene oxide, e.g. polyoxyethylene sorbitanmonooleate (Tween).

Examples of hydrophobic or water-emulsifying ointment bases areparaffins, vegetable oils, animal fats, synthetic glycerides, waxes,lanolin, and liquid polyalkylsiloxanes. Examples of hydrophilic ointmentbases are solid macrogols (polyethylene glycols). Other examples ofointment bases are triethanolamine soaps, sulphated fatty alcohol andpolysorbates.

Examples of other excipients are polymers such as carmelose, sodiumcarmelose, hydroxypropylmethylcellulose, hydroxyethylcel lulose,hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acaciagum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates,cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates andchitosans.

It is normally preferred that a local effect is obtained for thepolyclonal antibody, Clearance and thereby activity can be substantiallycontrolled and prolonged by pharmaceutical compositions such asmicrospheres, liposomes, complexes of positively or negatively chargedexcipients with antibody molecules of opposite charge.

Therapeutic Uses of Polyclonal Antibodies

In a preferred embodiment, the symphobody included in the presentcomposition is one that reacts with/binds to an inhalant allergenincluding conjunctival and nasopharyngeal allergens, as well asallergens entering the respiratory tract, or otherwise enters into thebody. The preventive or therapeutic inhalation of polyclonal antibodies,e.g. symphobodies, directed against common inhalant allergens is aimeddirectly at eliminating the cause of the allergy by aiding the blocking,neutralization, and clearance from the respiratory tract of the allergiccausative agent before allergic sensitization ensues.

Thus, the present embodiment of the invention concerns the possibilityof neutralizing the effect of allergen inhalation via polyclonalantibody inhalations by blocking allergen epitopes otherwise availablefor the binding of IgE molecules. Also, the binding of polyclonalantibodies is predicted to exert a clearance effect on allergens bymediating the phagocytosis and degradation of allergens without theinduction of allergic responses, as well as facilitating the upwardsclearance away from the respiratory tract into the pharynx of allergenentrapped in immune complexes with IgA or IgG together with mucosalmucous, and subsequent swallowing into the digestive tract.

Finally, the mucosal administration of allergen-specific polyclonalantibody, e.g. symphobody of the IgG or IgA isotype, which are blockingwith respect to the binding of allergen-specific IgE, is hypothesized toinhibit the IgE-mediated antigen presentation for T lymphocytes whichmay induce the predominantly T_(H)2 type T lymphocyte response toallergens which in allergic individuals is believed to perpetuate theallergy. Instead, the presence of blocking allergen-specific polyclonalantibodies, e.g. symphobodies may result in IgG- or IgA-mediated antigenpresentation for T cells, which in turn may preferentially promote aT_(H)1 type T lymphocyte response to allergens, thus interrupting thevicious cycle of the allergic inflammatory reaction.

Allergen epitopes e.g. from pollen are derived from several proteins,and thus for a single inhalant antibody to be able to work, it will berequired to contain several if not many individual idiotypicspecificities/antigen reactivities. In this respect, polyclonalantibodies seem far superior to monoclonal antibodies.

Consequently, polyclonal antibody compositions may be used for theprophylaxis or treatment of all types of allergy, including allergicrhinitis, hay fever, allergic conjunctivitis, and allergic (extrinsic)asthma, as well as food allergy. In particular, but not limited to, thepolyclonal antibody of the present invention is one that reactswith/binds to an allergen from: The house dust mites (e.g.Dermatophagoides farinae or D. pteronyssimus); danders from cat, dog, orhorse; tree pollens from birch (Betula alba), alder, hazel, oak, willow,plane, beech, elm, maple, ash, and hornbeam; grass pollens from timothygrass (Phleum pretense), bluegrass (Poa pratense), rye grass (Loliumperenne), Orchard grass (Dactylis glomerata), ragweed (e.g. Ambrosiaartemisiifolia), sweet vernal grass (anthoxanthum odoratum), and rye(Secale cereale); or fungi (e.g. Alternaria, Aspergillus, Cladosporium,and Penicillium). In addition, allergen-specific polyclonal antibodies,e.g. symphobodies may be used to treat allergies against other agentssuch as food allergens (e.g. peanuts and other nuts, shell-fish, egg,milk, corn) or bee venom allergens. Many of these allergens may bepurchased as well-characterized proteins from commercial suppliers.

The dose of polyclonal antibody required in humans to be effective inthe treatment or prevention of allergy differs with the type andseverity of the allergic condition to be treated, the type of allergen,the age and condition of the patient, etc. Typical doses of polyclonalantibody to be administered are in the range of 1 μg to 1 g, preferably1-1000 μg, more preferably 2-500, even more preferably 5-50, mostpreferably 10-20 μg per unit dosage form.

Experimental

The present invention is described in detail in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

Immunization of Mice for the Generation of Symphobody Libraries

BALB/c mice are immunized subcutaneously (s.c.) or intraperitoneally(i.p.) with e.g. 1 mg of allergenic protein in Freunds completeadjuvant. Immunization is performed using recombinant allergen protein(e.g. Der p 1) or extracts from native allergens. Any subsequentimmunizations are given at two to three week intervals and in incompleteFreunds adjuvant. Spleen and/or bone marrow are taken 3 days after thelast immunization and used for the preparation of the symphobodylibrary, as described in U.S. Pat. No. 5,789,208.

Generation of Symphobody Libraries from Allergic Patient Material

Symphobody libraries are prepared from blood or bone marrow samplestaken from allergic patients characterized by positive case history,skin prick testing, radioallergosorbent test (RAST), or reactivity ofpatient sera with allergen extracts by IgG or IgE immunoblotting orreactivity to purified recombinant allergens (e.g. pollen allergens oranimal allergens).

Antibody Binding to Allergen is Detected By ELISA

Between 50 and 1000 ng of allergen, disintegrated allergen, orrecombinant allergen are coated pr well of Nunc Maxisorp 96-wellmicrotiter plates, After washes in PBS containing gelatin or BSA as wellas Tween-20 the wells are blocked 1 hour at 37° C. using gelatin or BSA.Subsequently the wells are washed and incubated with either polyclonalantibodies, e.g. symphobodies, murine or human IgE, IgG derived fromeither serum or bronchoalveolar lavages (BAL). After repeated rounds ofwashing, bound antibody is detected by successive incubations ofsecondary biotinylated anti-mouse or anti-human immunoglobulin asappropriate, followed by AP-avidin, and pNPP substrate. Previouslycharacterized allergen-specific monoclonal antibodies are used as apositive control and monoclonal and polyclonal antibodies withdifferent, unrelated specificities are used as negative controls.

In some experiments polyclonal antibody incubations are preceded byincubations with well-characterized monoclonal antibodies in acompetitive ELISA.

Polyclonal Antibody Inhibition of Binding of Patient-Derived IgE toAllergens

Patient-derived IgE binding to allergen extracts is studied either incompetitive ELISA (similar to the protocol above with the followingmodifications) for IgE binding or by preparative SDS-PAGE and Westernblotting. After ELISA well coating or allergen electrophoresis usingallergen, disintegrated allergen, or recombinant allergen, theallergen-coated surface is blocked with gelatin or BSA, beforeincubation 3-4 hours at 4° C. with allergen-specific polyclonalantibodies. Subsequently, samples are incubated 3-4 hours at 4° C. withpatient sera or BAL IgE diluted 1:5 and bound human IgE antibodies aredetected with e.g. ¹²⁵I-labeled anti-human IgE antibodies (RAST;Pharmacia) and visualized by autoradiography. Binding of mouse IgG isdetected as described above.

Characterization of Polyclonal Antibody Reactivity with AllergenExtracts By Electrophoresis and Western Blotting

Allergen extracts are separated by SDS-PAGE and immunoblotted ontonitrocellulose strips before incubation with the antibody preparation(patient sera, mouse sera, polyclonal antibodies, e.g. symphobodies, orcontrol monoclonal antibodies). In some experiments, thecross-reactivity of polyclonal antibodies generated against one allergenis examined by testing in ELISA or Western blotting against a panel ofhomologous allergens.

Inhibition of Allergen-Induced Histamine Release from Human BasophileGranulocytes After Preincubation of Allergens with Polyclonal Antibodies

Heparinized blood samples are obtained from allergic patients andgranulocytes isolated by dextran sedimentation. Recombinant allergens,disintegrated allergens or allergen extracts are preincubated withallergen-specific polyclonal antibodies, e.g. symphobodies, or controlantibodies or buffer alone, for 1 h at room temperature beforeincubation at different concentrations (1, 0.1, 0.01, and 0.001 μg/ml)with granulocytes disintegrated in histamine release buffer (20 mMPIPES, pH 7.4, 110 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 g/L glucose, 0.3mg/ml human serum albumin). Histamine release into the cell-freesupernatant is determined by radioimmunoassay and expressed as apercentage of total histamine release after cell lysis.

Polyclonal Antibody Inhibition of Allergic Inflammation in a Mouse Modelof Allergy

Mice (e.g. BALB/c mice, are sensitized to allergens (e.g. ragweedallergen) by two or more i.p. injections of allergen (e.g. 150microgram) and alum on e.g. days 0 and 4. On e.g. day 11 and in a two tofour week timespan, an intratracheal or intranasal allergen challenge isperformed on anesthetized mice where after mice are analyzed asdescribed below. In some experiments a mouse model based on ovalbumin(OVA)-sensitization is employed. Briefly, BALB/c mice are injected i.p.with e.g. 5-100 μg OVA (chicken egg albumin grade V,Sigma) in 2 mgaluminum hydroxide adjuvant (alum, Pierce) on day 1 and day 14, beforechallenge on protocol days 28, 29 and 30 with either 1% aerosolized OVAin PBS for 20 minutes using a ultrasonic nebulizer (DeVilbiss Somerset,Pa, USA) or 5-100 μg OVA in 40 μL PBS injected intratracheally inanesthetized mice. Control mice receive the same amount of PBS.

On day 32, 24 hours after antigen challenge, 12 mice are subjected to anairway responsiveness test and killed on day 33. In the control group 8mice are used.

The left lung is tied of and BAL of the right lung is obtained by 5repeated washings with 200 μL PBS. The left lung is fixed and embeddedin paraffin for lung histology. A blood sample (tail blood) is alsotaken from each mouse and stored at −80° C. until analysis is carriedout.

In experiments where the ability of allergen-specific polyclonalantibodies, e.g. symphobodies to inhibit allergic inflammation isexamined, the allergen-specific polyclonal antibody preparationin dosesvarying from 1 μg to 1 mg is administered before, during, or after theadministration of the challenge dose of antigen.

Polyclonal antibodies with different or unrelated specificities as wellas PBS is used as a negative control, and the effect is in someexperiments compared with a positive control allergen-specificmonoclonal antibody.

Efficacy Evaluation of Polyclonal Antibodies in Blocking the AllergicResponse in the Murine Allergy Model

Upon completion of the allergen challenge, the allergic reaction isevaluated by performing bronchial lavage (BAL) on euthanized mice, andthe BAL fluid is examined by differential counting for the content ofeosinophils, neutrophils, lymphocytes, and macrophages.

The lower and upper lobes of the left lung are collected and fixed inCarnoy's solution (6× ethanol; 3× acetic acid glacial; 1× chloroform) at20° C. for ˜15 hours. After embedding in paraffin the tissues are cutinto 4-5 μm sections. From each mouse 10 airway sections randomlydistributed are assessed for severity of the cellular inflammation andmucus occlusion. The cellular infiltrate of the peribronchial andperivascular areas is evaluated semi-quantitatively for the presence ofleukocytes (eosinophils, iymphocytes), quantified on a scale from 0-5with an increment of 0.5. Mucus occlusion of the bronchial lumen isassigned a score using the following measures 0, 0-10% occlusion; 1,10-30% occlusion; 2, 30-60% occlusion; 3, 60-90% occlusion; 4, 90-100%occlusion. Damage to the airway epithelium is also estimated on anequivalent scale. All evaluations are performed by individuals blindedto the protocol design and the results are recorded photographically.The tissue sections are stained with hematoxylin and eosin for cellularstaining or hematoxylin and periodic acid-Schiff for mucus staining.

Total and OVA-specific IgE, IgG, IgG₁, IgG_(2a) and IgG₃ levels in theblood of mice are determined by ELISA as described above.

1. A pharmaceutical composition comprising as an active ingredient arecombinant polyclonal antibody or a mixture of individual monoclonalantibodies or an isolated or purified polyclonal antibody capable ofreacting with or binding to an allergen together with one or morepharmaceutically acceptable excipients.
 2. The pharmaceuticalcomposition of claim 1, wherein the active ingredient is a recombinantpolyclonal antibody.
 3. The pharmaceutical composition of claim 1,wherein the active ingredient is a mixture of individual monoclonalantibodies.
 4. The pharmaceutical composition of claim 1, wherein theactive ingredient is an isolated or purified polyclonal antibody.
 5. Thepharmaceutical composition of claim 1, where the composition is free ofthe allergen to which the antibody is reactive or binds.
 6. Thepharmaceutical composition of claim 1, wherein the composition comprisesat least one pharmaceutically acceptable excipient capable of effectingtopical application of said recombinant polyclonal antibody or saidmixture of individual monoclonal antibodies or said isolated or purifiedpolyclonal antibody.
 7. The pharmaceutical composition of claim 5,wherein said composition is intended for topical administration to theoropharynx, nasal cavity, respiratory tract, gastrointestinal tract,conjunctival mucosa, vagina, urogenital mucosa, or for dermalapplication.
 8. The pharmaceutical composition of claim 7, wherein therespiratory tract comprises the nasal, oral, pharyngeal, bronchial oralveolar mucosa.
 9. The pharmaceutical composition of claim 1, whereinsaid composition is provided as a solution, dispersion, powder, or inthe form of microspheres.
 10. The pharmaceutical composition of claim 2,wherein the recombinant polyclonal antibody is generated by phagedisplay technology.
 11. The pharmaceutical composition of claim 10,wherein the recombinant polyclonal antibody is generated under suchconditions that the immunoglobulin heavy chain variable region and lightchain variable region gene segments are linked together in a parentallibrary in order to allow for the bulk transfer of variable region lightchain and heavy chain gene pairs from one vector to another, whileallowing stable pairing of specific immunoglobulin variable region lightchain and heavy chain gene segments as they are present upon selectionfrom the parental library of immunoglobulin variable region light chainand heavy chain gene segment pairs encoding antibody molecules capableof reacting with or binding to an allergen.
 12. The pharmaceuticalcomposition of claim 10, wherein the recombinant polyclonal antibody isgenerated under such conditions that the immunoglobulin heavy chainvariable region and light chain variable region gene segments are linkedtogether in order to allow for the bulk transfer of specific variableregion light chain and heavy chain gene pairs from one vector toanother, while allowing stable pairing of specific immunoglobulinvariable region light chain and heavy chain gene segments as they arepresent in the original polyclonal immune response of an animal or humanindividual.
 13. The pharmaceutical composition of claim 1, wherein theallergen is an allergen of a house dust mite, dander from a cat, a dog,or a horse, tree pollen, grass pollen, or fungi.
 14. The pharmaceuticalcomposition of claim 1, wherein the recombinant polyclonal antibody orthe mixture of monoclonal antibodies or the isolated or purifiedpolyclonal antibody is provided in an amount in the range of 1 μg to 1 gper unit dosage form.
 15. The pharmaceutical composition of claim 14,wherein the recombinant polyclonal antibody or the mixture of monoclonalantibodies or the isolated or purified polyclonal antibody is providedin an amount in the range of 1 μg to 1000 μg per unit dosage form.
 16. Amethod of preventing or treating allergy, said method comprisingadministering to a patient in need thereof a pharmaceutical compositioncomprising as an active ingredient a recombinant polyclonal antibody ora mixture of individual monoclonal antibodies or an isolated or purifiedpolyclonal antibody capable of reacting with or binding to an allergento which the patient has shown or is predisposed to develop an allergicreactions together with one or more pharmaceutically acceptableexcipients.
 17. A method of inducing tolerance to an allergen, saidmethod comprising administering to a patient, who if untreated would belikely to show allergic reaction to the allergen, a compositioncomprising as an active ingredient a recombinant polyclonal antibody ora mixture of individual monoclonal antibodies or an isolated or purifiedpolyclonal antibody capable of reacting with or binding to an allergenand inducing tolerance to the allergen in the patient.